Effects of Pinacidil on Refractoriness in Acutely Ischemic Tissue: Insights from Experiments and Simulations

B Trenor1, JM Ferrero (Jr)1, J Saiz1, A Ferrero2, J Chorro2, M Monserrat1, V Hernández3, G Moltó3

1Center for Research and Innovation in Bioengineering, Universidad Politecnica de Valencia, Spain 2Servicio de Cardiologia, Hospital Clinico Universitario de Valencia, Spain 3Grupo de Redes y Computación de Altas Prestaciones, Universidad Politecnica de Valencia, Spain

and hypoxia provoke important changes in the electrical Abstract activity of the ischemic cells, creating inhomogeneities A controversy exits regarding the arrhythmogenic within the tissue and thus setting the stage for reentry. effect of potassium channel openers, such as pinacidil, Special attention must be paid to the action potential during acute myocardial ischemia. In order to throw duration (APD) reduction and refractoriness changes some insight into this issue, the effect of pinacidil on the provoked by ATP-sensitive potassium channels (K(ATP) refractoriness of normal and acutely ischemic tissue has channels) activation. These effects concomittant with been experimentally and theoretically studied. conduction velocity alteration are responsible, in part, for Experiments with Langendorff perfused rabbit hearts reentry generation and maintenance [6]. were conducted, as well as computer simulations based As pinacidil enhances ATP-sensitive potassium current on Luo-Rudy phase II model of the cardiac action (IK(ATP)) [7], it is relevant to study the effect of this drug potential. Our results show that pinacidil significantly on APD and effective refractory period (ERP) in order to reduces the effective refractory period (ERP) in normal evaluate possible actions on arrhythmogenic episodes tissue. However, in spite of strongly reducing QT interval, related to myocardial ischemia. In normoxia, pinacidil pinacidil does not have a significant effect on ERP in significantly reduces ERP [8,9], however these changes acutely ischemic tissue. In conclusion, during myocardial under acute myocardial ischemia are yet to be addressed. ischemia, despite action potential duration (APD) The goal of this study was to analyze experimentally reduction provoked by pinacidil, ERP is not significantly and theoretically, by means of computer simulation, modified by the drug due to the counteracting effect of changes in APD and ERP under normal and acute IK(ATP) on depolarization currents ischemic circumstances in the presence of pinacidil. 2. Methods 1. Introduction 2.1. Experimental study Potassium channel openers, such as pinacidil, are known to be vasodilatory drugs with controversial effects Fourteen New Zealand rabbits were anesthetized with on arrhythmogenic processes. Many experimental studies intravenous ketamine and then heparinized. The heart was addressing the behavior of this drug suggest that pinacidil immediately removed and immersed in cold (4ºC) Tyrode increases the potential for the development of ventricular solution. After isolation, the aorta was connected to a fibrillation under different circumstances, such as Langendorff system for perfusion with Tyrode solution at myocardial ischemia or hypoxia [1-3]. On the contrary, a temperature of 37ºC. The heart was oxygenated with a other authors sustain the hypothesis of the dose- mixture of 95% O2 and 5% CO2. The ventricles were dependent antiarrhythmic effect of pinacidil [4,5], electrically stimulated by means of a bipolar electrode especially in arhythmogenic episodes related to triggered using a GRASS S800 (Grass Instruments Inc.) activity. programmable stimulator. A train of basic stimuli with a Focussing the effect of pinacidil on arrhythmogenic basic cycle length (BCL) of 250 ms with delivered prior processes related to reentrant activity and arising during to the test stimulus which was used to determine the ERP. acute myocardial ischemia, it is important to understand Electrograms were recorded by a multi-electrode array of the electrophysiological changes that take place under 256 electrodes (1mm interelectrode distance). In the such circumstances. Indeed, during myocardial ischemia pinacidil group, 10 µmol/L of pinacidil was administrated metabolic impairment, potassium accumulation, acidosis

0276−6547/04 $20.00 © 2004 IEEE 529 Computers in Cardiology 2004;31:529−532. by dissolving rthe drug in the Tyrode solution. In the under different situations. The results were compared and ischemia group, the circumflex artery was ligated and are depicted in Figure 2. In analyzing the effects of recordings were taken 5 minutes after artery ligation. ischemia on the ERP, we compared control ERPs (first and third bars) with ischemic ERPs (second and fourth 2.2. Simulation study bars), in the absence (first two bars) and presence of 10 µmol/L pinacidil (second two bars). ERP values were In order to simulate the electrical activity of ventricular normalized to the control value in the absence of the drug. cells we used a modified version of Luo-Rudy Phase II A significative decrease of ERP was observed in the action potential (AP) model [10] [11], in which absence of the drug, however the difference was non- IK(ATP)current formulation [12] and the action of pinacidil signiticative (NS) in the presence of pinacidil. on this current [13] were included. As for the effects of pinacidil on ERP, normal cells We considered a 1D strand of 200 cells and recovered excitability before in the presence of pinacidil propagation of action potential was studied. Firstly, (bar six) than in its absence (bar five), and the difference normal physiological conditions were considered, and was significative. On the contrary, this difference was APD and ERP were measured in the absence and non-significative (NS) under ischemic conditions (two presence of 10 µmol/L of pinacidil. Secondly, last bars). This last observation determined the second physiological conditions were altered so as to reproduce part of our study. an acute ischemic episode. Indeed, metabolical impairment was mimiqued by reducing intracellular ATP Normalized ERP (ms) 120 concentration from normoxic values (6.8 mmol/L) to 4.4 NS p NS mmol/L and increasing intracellular ADP concentration 100 from typical normoxic values (15 mmol/L) to 150 i p mmol/L [14]. Acidosis, which also arises during 80 i myocardial ischemia, is known to block partially sodium 60 and calcium channels [15-17], and thus these currents were multiplied by 0.9. Finally, extracellular potassium 40 concentration was increased from normoxic values (5.4 20 mmol/L) to ischemic values (11 mmol/L) [14]. Under these circumstances, APD and ERP were also measured 0 in the absence and presence of 10 µmol/L of pinacidil. [P]=0 [P]=10 CONTROL ISCHEMIA The protocol of stimulation is depicted in figure 1. A Figure 2. Experimental measured and normalized premature current pulse was applied at different coupling ERPs. C stands for control, i for ischemia and P for intervals (CI) after the basic stimulus given at t=50 ms. pinacidil. Their amplitudes were 1.5 times diastolic threshold and the duration was 2 ms. In second place, computer simulations were conducted in order to throw some insight into the mechanism of Basic Premature action of pinacidil. The above described experiments were simulated and similar results were obtained. Pinacidil strongly reduced APD under ischemic 1.5 × Ith conditions, as shown in Figure 3. A less important decrease was also observed in normoxia (results not 2 ms CI (ms) shown). As expected, also ischemia shortened APD in the t0=50 ms presence, as well as in the absence of the drug. Pinacidil and ischemia also reduced ERP as shown in Figure 1. Stimulation protocol. table 1.

Both current pulses were applied to one edge of the Table 1. Simulated ERP in milliseconds strand. The ERP was measured as the shortest CI leading Control Ischemia to AP propagation. APD was measured at 90% of [P]=0 180 147 repolarization. [P]=10 µmol/L 161 144

3. Results From the obtained results, it can be deduced that the In fist place, the ERP was experimentally measured reduction in ERP provoked by pinacidil was less

530 important under ischemic conditions, results also has generated extensive data on APD and ERP shortening observed experimentally. 10 [P]= IK(ATP) (µA/cm2) Action Potential (mV) 40 [P]=0 [P]=0 20 [P]=10µmol/L 5 0

-20

-40 0 220 240 260 280 300 320 -60 t (ms) 0 -80 50 100 150 200 250 300 -20 [P]= 10µmol/L t (ms) -40 [P]=0 -60 Figure 3. Basic and first propagated premature action potentials in the absence and presence of 10 µmol/L -80 2 pinacidil, under ischemic conditions. -100 INa (µA/cm )

Our analysis was then foccussed on trying to Figure 4. IKATP and INa currents during ischemia in the understand this effect. absence and in the presence of 10 µmol/L pinacidil, after As depicted in Figure 3, allthough APD was strongly the premature stimulus. reduced by pinacidil under ischemia (from 72 milliseconds in the absence of the drug to 45 milliseconds by pinacidil under normal physiological conditions in the presence of pinacidil), reduction of ERP was not so [8,20]. Our experimental and simulation results are in remarkable. Indeed, in the absence of the drug, a CI of accordance with these observations. 147 milliseconds led to AP propagation, whereas under If we consider the effects of the drug on ERP under the effects of pinacidil a CI of 144 milliseconds led to AP pathological situations, such as the acute phase of propagation. Postrepolarization refractoriness was then 24 ischemia, little experimental or theoretical results have milliseconds longer in the second case. been documented. The present study suggests that the Ionic currents were also measured under these difference between ERP reduction caused by pinacidil ischemic conditions in order to analyze the effect of the under normoxia and under ischemia is non-significative. drug. Figure 4 shows the time course of IK(ATP) outward Our experimental conditions were mimicked by computer current and inward sodium current (INa) in both cases. The simulations in order to investigate ionic mechanisms enhanced activity of K(ATP) channels in ischemia is associated with pinacidil effects on refractoriness. As further increased by pinacidil. And finally, the inward related in the previous section, during ischemia, sodium current must reach a higher level in the presence postrepolarization refractoriness was longer in the of the drug to allow AP propagation. presence of the drug. Indeed, in the presence of the drug APD shortening was greater than ERP shortening. A 4. Discussion and conclusions shorter APD in the presence of pinacidil allows a sooner Several experimental studies have pointed out the dose recuperation of refractoriness, so as to expect a dependent effects of pinacidil on arrhythmias significantly shorter ERP. However, an enhanced IK(ATP) [1,3,4,18,19]. Particularly, reentrant arrhythmias arising current under the effects of pinacidil (see Figure 4), during the acute phase of myocardial ischemia are counteracts depolarizing sodium current, and thus INa subjected to the effect of this drug. Under such must reach higher values (see Figure 4) to assure action circumstances, much attention should be paid to potential upstroke and propagation. Consequently, CI electophysiological alterations related to refractoriness must be longer enough to assure sodium channels and APD, which are involved in reentrant circuit availability, so that ERP is not significantly reduced. generation and maintenance [6]. The above described effects exerted by pinacidil on This study focussed on APD and ERP alterations ERP can have important consequences on caused by pinacidil under the acute phase of myocardial arrhythmogenesis. Moreover its intricate mechanism of ischemia or normoxia. The broad experimental literature action colud respond for the existent controversy in the experimental litterature, concerning the proarrhythmic

531 action of this drug. Two opposite effects in terms of [9] Friedrichs GS, Chi L, Black SC, Manley PJ, Lucchesi BR. refractoriness must be taken into account. On the one Antiarrhythmic agent, MS-551, protects against pinacidil + hypoxia- induced ventricular fibrillation in Langendorff- hand, the activation of K(ATP) channels provoked by pinacidil, reduces significantly APD, so that the recovery perfused rabbit isolated heart. J. Cardiovasc. Pharmacol. 1994; 23: 120-126. of excitability starts before reducing ERP. On the other [10] Luo CH, Rudy Y. A dynamic model of the cardiac hand, the enhanced activity of IK(ATP) counteracts ventricular action potential. I. Simulations of ionic currents depolarization currents so that postrepolarization and concentration changes. Circ. Res. 1994; 74: 1071-96. refractoriness can be increased. The administered dose of [11] Zeng J, Laurita KR, Rosenbaum DS, Rudy Y. Two the drug is then determinant to evaluate its net effect on components of the delayed rectifier K+ current in refractoriness and thus on arrhythmogenic activity related ventricular myocytes of the guinea pig type. Theoretical to reentrant circuits. Also experimental and physiological formulation and their role in repolarization. Circ. Res. conditions are extremely important in determining the 1995; 77: 140-152. action exerted by the drug. [12] Ferrero JM, Jr., Saiz J, Ferrero JM, Thakor NV. Simulation of action potentials from metabolically impaired cardiac Acknowledgements myocytes. Role of ATP-sensitive K+ current. Circ. Res. 1996; 79: 208-221. This work was partially supported by (1) the Plan [13] Trenor B, Ferrero JM, Jr., Rodriguez B, Saiz J, Ferrero JM, Nacional de Investigación Científica, Desarrollo e Thakor N. Simulation Study of the Effect of Pinacidil on Innovación Tecnológica del Ministerio de Ciencia y ATP-Sensitive Potassium Current and Action Potential Tecnología of Spain (TIC 2001-2686) and (2) the Duration in Myocardial Tissue. Computers in Cardiology Programa Incentivo a la Investigación de la Universidad 1998; 25:581-584. [14] Weiss JN, Venkatesh N, Lamp ST. ATP-sensitive K+ Politecnica de Valencia (CAMAV Project 20020418). channels and cellular K+ loss in hypoxic and ischaemic References mammalian ventricle. J. Physiol 1992; 447: 649-673. [15] Watson CL, Gold MR. Effect of intracellular and [1] Wolleben CD, Sanguinetti MC, Siegl PK. Influence of extracellular acidosis on sodium current in ventricular ATP-sensitive potassium channel modulators on ischemia- myocytes. Am. J. Physiol 1995; 268: H1749-H1756. induced fibrillation in isolated rat hearts. J. Mol. Cell [16] Shaw RM, Rudy Y. Electrophysiologic effects of acute Cardiol. 1989; 21: 783-788. myocardial ischemia. A mechanistic investigation of action [2] Chi L, Uprichard AC, Lucchesi BR. Profibrillatory actions potential conduction and conduction failure. Circ. Res. of pinacidil in a conscious canine model of sudden 1997; 80: 124-138. coronary death. J. Cardiovasc. Pharmacol. 1990; 15: 452- [17] Kagiyama Y, Hill JL, Gettes LS. Interaction of acidosis 464. and increased extracellular potassium on action potential [3] D'Alonzo AJ, Zhu JL, Darbenzio RB, Dorso CR, Grover characteristics and conduction in guinea pig ventricular GJ. Proarrhythmic effects of pinacidil are partially muscle. Circ. Res. 1982; 51: 614-623. mediated through enhancement of catecholamine release in [18] Padrini R, Bova S, Cargnelli G, Piovan D, Ferrari M. isolated perfused guinea-pig hearts. J. Mol. Cell Cardiol. Effects of pinacidil on guinea-pig isolated perfused heart 1998; 30: 415-423. with particular reference to the proarrhythmic effect. Br. J. [4] D'Alonzo AJ, Darbenzio RB, Hess TA, Sewter JC, Sleph Pharmacol. 1992; 105: 715-719. PG, Grover GJ. Effect of potassium on the action of the [19] Lepran I, Baczko I, Varro A, Papp JG. ATP-sensitive KATP modulators cromakalim, pinacidil, or glibenclamide potassium channel modulators: both pinacidil and on arrhythmias in isolated perfused rat heart subjected to glibenclamide produce antiarrhythmic activity during acute regional ischaemia. Cardiovasc. Res. 1994; 28: 881-887. myocardial infarction in conscious rats. J. Pharmacol. Exp. [5] Carlsson L, Abrahamsson C, Drews L, Duker G. Ther. 1996; 277: 1215-1220. Antiarrhythmic effects of potassium channel openers in [20] Friedrichs GS, Chi L, Black SC, Manley PJ, Lucchesi BR. rhythm abnormalities related to delayed repolarization. Antiarrhythmic agent, MS-551, protects against pinacidil + Circulation 1992; 85: 1491-1500. hypoxia-induced ventricular fibrillation in Langendorff- [6] Nassif G, Dillon SM, Rayhill S, Wit AL. Reentrant circuits perfused rabbit isolated heart. J. Cardiovasc. Pharmacol. and the effects of heptanol in a rabbit model of infarction 1994; 23: 120-126. with a uniform anisotropic epicardial border zone. J. Cardiovasc. Electrophysiol. 1993; 4: 112-133. Address for correspondence. [7] Fan Z, Nakayama K, Hiraoka M. Multiple actions of Beatriz Trenor. pinacidil on adenosine triphosphate-sensitive potassium Universidad Politecnica de Valencia channels in guinea-pig ventricular myocytes. J. Physiol Camino de Vera s/n Valencia 46022. Spain. 1990; 430: 273-295. [email protected] [8] Sanguinetti MC, Scott AL, Zingaro GJ, Siegl PK. BRL 34915 (cromakalim) activates ATP-sensitive K+ current in cardiac muscle. Proc. Natl. Acad. Sci. U. S. A 1988; 85: 8360-8364.

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